Herbaceous plant species Chenopodium ambrosioides has natural compounds with anti-androgenic qualities and can be utilized as a contraceptive source. The goal of the current study was to investigate the antifertility activity of C. ambrosioides leaf extract in adult male rats using phytochemical analysis. Total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity of the methanolic extract were assessed. Additionally, GC-MS analysis was performed to closely examine the extract’s bioactive phyto-constituents. An in vitro experimental approach was employed to evaluate the antifertility action by directly observing the impact of varying amounts of plant extract on the integrity of sperm DNA, the antioxidant state, and the release of testosterone in the testis. Results showed presence of adequate amount of total flavonoid and phenolic contents along with antioxidant potential. A GC-MS investigation of the methanolic leaf extract revealed that C. ambroisoides contains thirteen bioactive components. The maximum dose regimen (1000 \(mu\)g/mL) treatment in the in vitro experiment showed significantly higher oxidative stress and decreased antioxidant activity. The rat sperm experience DNA damage as a result of the elevated ROS and lipid peroxidation concentration. Similarly, following two hours of incubation with all of the chosen dosages of C. ambrosioides leaf extract, a decrease in the levels of testosterone in the testicles was observed. According to the current study’s findings, C. ambrosioides may affect male fertility by causing oxidative stress, hormonal imbalances, and sperm DNA integrity disruption in rat testicles exposed to greater extract concentrations.